Recently, demands for the development of technology used for analyzing the activity of physiological materials, such as nucleic acids, proteins, enzymes, antibodies, and antigens have rapidly increased in the world. For such demands, a biochip in which the required physiological material molecules are immobilized on certain tiny regions by adopting semiconductor processing techniques is suggested, so thereby physiologically useful information is easily obtained just by biochemically searching the biochip.
The biochip is in the form of a conventional semiconductor chip, but what is integrated thereon is a bio-organic material such as an enzyme, a protein, an antibody, DNA, a microorganism, animal and plant cells and organs, a neuron, and so on. The biochip can be classified into a “DNA chip” immobilizing a DNA probe, a “protein chip” immobilizing a protein such as an enzyme, an antibody, an antigen and so on, and a “lab-on-a-chip” which is integrated with pre-treating, biochemical reacting, detecting, and data-analyzing functions to impart an auto-analysis function.
To achieve the successful development of such a biochip, it is important to find a method for immobilizing a physiological material in which an interface between the physiological material and a substrate is efficiently formed, and the inherent functions of the physiological material can be utilized at a maximum level. Generally, the physiological material is immobilized on the surface of a glass slide, a silicon wafer, a microwell plate, a tube, a spherical bead, a surface with a porous layer, etc. by various techniques, for example, by reacting DNA with carbodiimide to activate a 5′-phosphate group of DNA, and by reacting the activated DNA with a functional group on the surface of the substrate so as to immobilize the DNA on the substrate.
U.S. Pat. No. 5,858,653 discloses a composition comprising an ion group, such as a quaternary ammonium group, a protonated tertiary amine, or phosphonium, capable of reacting with a target physiological material, and a polymer having a photo-reactive group or a thermochemically reactive group for use in attaching to the surface of substrate. U.S. Pat. No. 5,981,734 teaches that when DNA is immobilized by a polyacrylamide gel having an amino group or an aldehyde group, the DNA can be bound with a substrate via a stable hybridization bond to easily facilitate carrying out of analysis. U.S. Pat. No. 5,869,272 discloses an attachment layer comprising a chemical selected from dendrimers, star polymers, molecular self-assembling polymers, polymeric siloxanes, and film-forming latexes formed by spin-coating a silicone wafer with aminosilane. U.S. Pat. No. 5,869,272 also discloses a method for the determination of a bacteria antigen by detecting a visual color change of an optically active surface. U.S. Pat. No. 5,919,523 discloses a method for preparing a support on which an amino silane-treated substrate is doped with glycine or serine or is coated with an amine, imine, or amide-based organic polymer. U.S. Pat. No. 5,985,551 discloses a method for providing amino groups on a solid substrate by using a photolithography technique on the amino silane treated substrate, the method involving allotting hydrophilic functions on regions to immobilize DNA and fluorosiloxane hydrophobic functions on other regions so as to form a desirable patterned DNA spot on the substrate.
In the above-mentioned patents, the immobilization layer is provided by preparing a self-assembly monolayer of silane molecules. Preferably, the silane is aminoalkoxy silane since it does not produce acidic by-products, and it can provide a molecular layer having a functional group with a relatively high density. Although much research has advanced the obtainment of a uniform monolayer having high-density functional groups using aminoalkoxy silanes, an aminosilane monolayer having a functional group with a uniform and high density and shorter manufacturing time has not been achieved.
The performance of the biochip is also affected by the nature of the substrate used as a support. That is, in order to utilize a spectroscopy technique on the hybridization analysis of the very dense array, the substrate should be optically transparent and the bond between the surface of the substrate and the physiological material should be stable.
Generally, the immobilization substrate is prepared by coating a sodium lime glass with amino silane compounds. The sodium lime glass is, however, a low level glass containing more than about 12% sodium. The sodium present in such glass is easily deposited, resulting in the degeneration of the transparency of the glass and the dissociation of the siloxane bond or the bond between the glass and silane. The immobilization layer thereby looses binding strength to the substrate, and it becomes difficult to achieve a uniform density of functional groups.
To solve the problems, borosilicate or boroaluminosilicate glass is suggested in WO 99/40038, but it has the disadvantage of being too expensive.